CELLULAR IMMUNOLOGY 53, 246-256 (1980)

A Study of Schistosoma mansoni Reinfection in Thymectomized Rats

DONATO CIOLI,* WALTER MALoRNI,t CESARE DE M.mTrNo,t AND GUNTHER DENNERT$

*Laboratory of Cell Biology, 18lA Via Romagnosi, 00196 Rome, Italy; tRegina Elena Institute for Cancer Research, Rome, Italy; SThe Salk Institute, La Jolla, California 92112

Received June 26, 1979

Thymectomized irradiated rats were reconstituted with bone marrow from thymectomized donors subjected to chronic thoracic duct drainage. These animals (“B rats”) showed a profound suppression of T-cell activity in various in vifro and in vivo tests. B rats of the Fisher strain infected twice with S. mansoni did not exhibit any resistance to reinfection, while intact controls showed a significant reduction in the number of challenge worms. High levels of peripheral eosinophils were present in infected intact rats, while B rats showed only modest eosinophilia under the same conditions of infection. Granuloma formation and other cellular inflammatory reactions in the liver of infected animals were almost completely abolished in B rats.

INTRODUCTION

Laboratory rats infected for the first time with Schistosoma mansoni spontaneously eliminate a large majority of parasites between the fourth and the sixth week after infection (“self-cure”). In addition, the few surviving parasites appear stunted and are unable to complete their life cycle in the rat, as evidenced by a lack of mature eggs in the feces of this “nonpermissive” host (1). We have previously shown that immune mechanisms are involved-at least in part-in the phenomenon of adult worm elimination since in T-cell-deprived (“B”) rats worm burdens remain elevated for considerably longer periods of time than in intact controls (2). However, worm maturation and oviposition remain severely impaired in immunosuppressed rats, thus suggesting that nonimmune mechanisms are also responsible for the nonpermissive status of laboratory rats. In addition, the timing of self-cure has been shown to depend upon the age of the worm rather than upon the length of host exposure to parasites, a fact which is difficult to make compatible with an interpretation of adult worm elimination in immunological terms (3).

A second infection of previously exposed rats results in challenge worm yields which-if determined before spontaneous elimination occurs-are significantly lower than those observed in previously uninfected animals (“resistance to reinfection”) (4-6).

In the present study we have examined the resistance of B rats upon a second exposure to S. mansoni, with the aim of providing an answer to the following

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Schisfosoma REINFECTION IN THYMECTOMIZED RATS 247

questions: (i) is resistance to reinfection a totally T-cell-dependent immune phenomenon? (ii) Is any modification of resistance correlated with modifications in other parameters (eosinophils, tissue granulomatous reactions) which have been postulated to play a role in the mechanisms of resistance?

MATERIALS AND METHODS

Ruts. Female inbred WistarlFurth (W/Fu), Fisher (F-344), and AC1 rats were purchased from Microbiological Associates, Inc. (Bethesda, Md.). Female outbred Wistar rats were purchased from Nossan Farms (Milan, Italy). Animals were 5-6 weeks old at the beginning of each experiment. The procedures for the preparation of B rats have been previously described (2). Briefly, rats were thymectomized, irradiated (1000 R), and reconstituted with bone marrow cells prepared from thymec- tomized donors which had been subjected to thoracic duct drainage for 7 days.

Assay of spleen cells for immune functions in vitro. The tissue culture medium used for all experiments was RPM1 1640 (Gibso, Santa Clara, Calif.) containing 5 x 10e5 M mercaptoethanol and 5% selected FCS. Serum A 660320 (Gibso) was used. For assay of mitogen stimulation and the mixed lymphocyte reaction (MLR), spleen cells were suspended in tissue culture medium in a series of tubes (Falcon Plastics No. 2057). To duplicate cultures of 1 ml cell suspension phytohemagglutinin P (PHA; Wellcome Laboratories, Beckenham, England), concanavalin A (Con A; Miles-Yeda, Kankakee, Ill.), pokeweed mitogen (PWM; Gibco, Berkeley, Calif.), dextran sulfate (Dex; Sigma, St. Louis, MO.), or mitomycin C (Sigma, St. Louis, MO.) treated lymphoid cells were added. At 24-hr intervals thereafter, cell proliferation was assayed by pulse-labeling the cultures with 0.1 ml RPM1 1640 containing 0.5 pCi[3H]thymidine (specific activity 25 Ci/mmol), lop5 M unlabeled thymidine, and lo-” M fluorodeoxyuridine. After 20 hr, the cells were harvested on glass fiber filters and the incorporated radioactivity was counted in a scintillation counter.

The induction of cell-mediated cytotoxicity was assayed by culturing responder spleen cells (2.5 x 10’) in 20 ml medium in Falcon tissue culture bottles (No. 3013) with an equal number of BALB/c stimulator spleen cells (1000 R irradiated) for 5 days. Cytotoxic activity was assayed on 51Cr-labeled BALB/c S194 target cells in RPM1 1640 medium containing 10% FCS. The following equation was used to express cytotoxicity:

% cytotoxicity = (experimental 51Crrelease - spontaneous 51Cr release) x loo

(maximal 51Cr release - spontaneous 51Cr release)

Maximal release was determined by freezing and thawing the target cells three times. All these procedures have been previously described (7).

In Fig. 1 various immune reactivities of spleen cells of normal and thymectomized rats are plotted. It is seen that all T-cell-mediated responses, namely, proliferation in MLR and induction of cell-mediated killing as well as the proliferative responses to PHA and Con A are either completely or at least severely suppressed. Quite in contrast, mitogen responses to dextran sulfate and pokeweed mitogen appear to be normal in both batches of animals. These experiments demonstrate that the thymectomized animals used in the following experiments are indeed immunodeficient where their T-cell responses are concerned.

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Schisfosoma REINFECTION IN THYMECTOMIZED RATS 249

TABLE 1

Resistance to Reinfection in Various Strains of Rats

Time No. of intervals Worms recovered + SE (No. of rats)

cercariae (days) Primary Challenge

1st 2nd Ist- 2nd- infection Double infection Percentage

Rat strain inf. inf. 2nd pelf. alone infection alone resistance” P

WiFu 300 1200 43 27 7.8 2 3.4 102.7 2 7.8 101.3 -c 7.6 6 NS (intact) (4) (6) (7)

WiFu 300 1200 43 28 13.3 i 2.8 148.5 t 26.5 112.8 2 7.6 - NS (B rats) (7) (6) (6)

Wistar 300 1600 49 28 6.8 -c 2.4 46.6 2 3.2 105.7 k 11.1 63 <O.OOl outbred (4) (3 (6)

W/Fu 500 1200 47 26 3.5 2 0.8 38.7 f 5.3 59.9 k 9.6 41 -0.05 (intact) (8) (7) (8)

AC1 500 1500 47 28 7.6 2 1.2 78.7 2 5.5 172.1 k 15.5 59 10.01

(9) (3) (9)

Fisher 500 1400 46 26 14.8 -c 3.0 49.6 2 7.6 184.4 2 17.4 81 <O.OOl

(10) (10) (9)

” Percentage resistance = [A - (B - C)/A] x 100, where A = No. of worms from challenge infection alone, B = No. of worms from double infection, and C = No. of worms from primary infection alone.

Immune responses ofrars in vivo. The production of anti-SRBC plaque-forming cells (PFC) and of hemolytic and hemagglutinating antibodies was determined as previously described (2), 5 days after a single ip injection of lo9 SRBC. Antischistosome antibodies were assayed by indirect hemagglutination (IHA) as previously described.

Parasitological procedures. The origin and the maintenance of S. mansoni, as well as the procedures used for animal infection and worm recovery have been described elsewhere (2, 8).

Eosinophil counts. Blood samples were obtained from the tail between 12 noon and 3 PM. Eosinophils were counted in Discombe’s fluid in hemocytometer chambers.

Morphological studies. Samples of liver tissue were obtained immediately after perfusion of rats for worm recovery. Preliminary experiments had shown no significant morphologic changes between perfused and nonperfused liver sections. Tissue fragments were fixed in 1% glutaraldehyde and postfixed in osmium tetroxide. After dehydration in alcohol, the fragments were embedded in Epon 812. Light microscopy observations were performed on l-pm-thick sections stained with buffered toluidine blue (pH 8). The type of cells in granulomas was established from observations on 50 different images resulting from about 40 thin sections stained with uranyl acetate and lead hydroxide and examined with a Siemens Elmiskop 101 electron microscope.

RESULTS

Resistance to Reinfection in Various Strains of Rats

Since our previous experiments on the course of a primary S. mansoni infection in B rats had been carried out in Wistar/Furth rats, we decided to study resistance to

CIOLI ET AL.

reinfection in the same strain of animals. The results of an experiment planned for this purpose are summarized in the first two lines of Table 1 and show that no significant resistance could be obtained in either thymectomized or intact animals. On the other hand, a similar experiment carried out using outbred Wistar rats which had received the same number of primary infection cercariae (Table 1, third line) resulted in clearly significant resistance to reinfection. These results prompted us to further investigate the phenomenon by comparing the resistance to reinfection of various strains of rats in the same basic experiment. The data in the fourth line of Table 1 indicate that for intact W/Fu rats, infected initially with a larger number of cercariae than in the previous experiment (line l), the level of resistance was just at the threshold of statistical significance. Resistance was greater in AC1 rats and even more pronounced in Fisher rats infected with the same number of cercariae. It was therefore decided to study the effect of T-cell depletion in Fisher rats.

Resistance to Reinfection in B Rats

A group of B rats (Fisher strain) and a group of age-matched intact controls were given a primary infection with 500 cercariae. Six weeks later the same animals received a challenge infection with 1300 cercariae, together with groups of age-matched previously uninfected B and intact rats. All the animals were sacrificed 3 weeks after challenge, i.e., a time when primary infection worms could

TABLE 2

Resistance to Reinfection in B Rats (Fisher Strain)

Twice infected: 500 cercariae -------- 6 weeks -------- 1300 cercariae -------- 3 weeks -------- perfusion. Challenge controls: ________________________________________--- 1300 cercaeae ________ 3 weeks ________ perfusion.

Intact rats B rats

Twice Challenge Twice Challenge infected controls infected controls

No. of rats 11 9 7 5 No. of challenge worms

recovered 2 SE” 47.8 ” 5.3 169.9 + 32.9 227.9 2 32.2 249.8 + 54.8 No. of primary infection worms

recovered + SE” 5.0 + 1.5 - 18.7 + 4.2 -

Percentage resistance 72 9 P <O.OOl NS

Anti-SRBC responses PFCI lo6 cells 2,3 19 42 Hemolysin titer 15,096 71 Hemagglutinin titelb 68 9

Antischistosome antibodies’ (indirect hemagglutination)” 182 <2

o In twice-infected animals, 3-week-old challenge infection worms were distinguished from primary- infection (9-week-old) worms on the basis of their size.

b Geometric means for hemagglutinin titers. r Only twice-infected animals.

Schisrosoma RE:NFECTION IN THYMECTOMIZED RATS 251

DAYS AFTER INFECTION

FIG. 2. Peripheral eosinophil levels of normal (0 - 0) and B rats (0 - - - 0) infected with S. mansoni. (A) W/Fu rats infected with 300 cercariae. (B) Fisher rats infected with 1000 cercariae.

be easily distinguished from challenge-derived schistosomes on the basis of their size. Five days before perfusion, all rats received IO9 SRBC ip in order to determine their humoral immune responsiveness. The results are presented in Table 2. While twice-infected intact rats exhibited a highly significant 72% reduction in challenge worm burdens with respect to previously uninfected controls, B rats showed an almost complete absence of resistance to reinfection. It should be noted that B rats were not resistant in spite of the fact that a larger number of primary-infection worms-presumably representing a larger antigenic stimulus-had survived in these immunosuppressed animals in comparison to those in intact controls, in accordance with our previous results (2). At 3 weeks after infection (a time point not studied previously) the number of challenge control worms was also higher in B rats than in intact animals (250 vs 170), but the difference was not statistically significant. All three assays used to evaluate anti-SRBC responses (PFC, HA, hemolysin) showed a drastic impairment of humoral immunity in B rats (Table 2). Similarly, antischistosome antibodies of twice-infected animals (assayed by passive HA) were reduced to undetectable levels in B rats.

252 CIOLI ET AL.

Peripheral Eosinophilia

The level of peripheral eosinophils was determined in two separate experiments. In the first experiment (Fig. 2A) a group of intact W/Fu rats and a group of B rats of the same strain were infected with 300 cercariae and the number of peripheral eosinophils was determined at weekly intervals. A similar experiment was also carried out in intact and B rats of the Fisher strain infected with 1000 cercariae, and the results are shown in Fig. 2B. In both experiments the number of peripheral eosinophils rose to rather high values (1500-2000 eosinophils/mm3) in intact rats, while B rats showed only a modest increase (up to about 500 eosinophils/mm3).

Liver Morphology

Representative liver sections from intact and B rats infected 10 weeks with 250 cercariae are shown in Fig. 3. Intact rats (Figs. 3A and B) presented a typical pattern of cellular infiltrates subverting the hepatic structure and large granulomas surrounding parasites and eggs. Cellular infiltrates consisted of eosinophils (39.9%), mast cells (31.8%), macrophages (12.5%), plasma cells (6.2%), lymphocytes (6.2%), a small amount of immature lymphocytes (2.5%), and neutrophils (0.9%). On the contrary, B rats (Figs. 3C and D) presented a well-preserved general hepatic structure, an absence of organized granulomas, and essentially no reaction even in close contact with parasites and eggs.

DISCUSSION

We have extended to twice-infected animals our previous analysis of S. mansoni infection in thymectomized rats (2). The experimental model we have adopted permits a particularly rigorous study of thymus-dependent phenomena, since “B rats” exhibit a very profound and specific chronic suppression of T-cell-mediated immune mechanisms, as shown by various in vitro and in vivo tests (Fig. 1 and Table 2).

The main finding of the present study is the demonstration that resistance to reinfection is completely abolished in B rats. This result strengthens previous observations pointing in various ways to immune mechanisms as the basis of resistance phenomena in the rat. Thus, peritoneal exudate cells which consist in part of T cells have been shown to mediate the adoptive transfer of resistance from 3-week-infected rats to normal recipients (9), while at later times (7 weeks after infection) a passive transfer of serum immunoglobulin is effective in conferring partial resistance (5). Knopfet al. (6), based on a study of different cercarial doses at challenge infection, concluded that resistance cannot be due to a nutritional limitation on worm survival (“athrepsia”), but rather to toxic factors produced by once-infected rats. Our results, showing a complete abrogation of resistance in thymectomized rats, support the concept that immune responses are an essential component of resistance to reinfection in this particular host.

The specific immune mechanisms leading to resistance to reinfection in schistosomiasis are currently the object of an active debate. It was of interest to examine in our system which of the phenomena presumably connected with resistance were modified in a situation where resistance was completely abolished. A possible key role for eosinophils in schistosome immunity has emerged in recent

Schistosomn REINFECTION IN THYMECTOMIZED RATS 253

years (for a review, see Ref. (10)). We have examined the pattern of peripheral eosinophilia in infected B rats and found in these immunosuppressed hosts a pronounced depression of the typical eosinophilic response shown by intact rats (11). This result is in accordance with the T dependence of eosinophilia demonstrated in T. @a/is-infected rats (12) and parallels a similar depression of peripheral eosinophilia following S. mansoni infection as observed in thymec- tomized mice (13) or in congenitally athymic mice (14, 15). Thus, it is clear that T-dependent effector molecules or stimuli have to play a role in eosinophilia, but no formal explanation for this effect has been given. Although the level of peripheral eosinophils was clearly depressed in B rats, their eosinophilic response was not as completely abolished as one would have expected from the dramatic suppression of resistance to reinfection. This may be tentatively taken as a suggestion that eosinophils per se (i.e., in the absence of T-cell-dependent products or reactions) are not sufficient to result in resistance to reinfection in the absence of other com- ponents of the immune response (e.g., antibody), in accordance with all previous observations (10, 16).

The scarcity of eosinophils in peripheral blood of B rats was also reflected in tissue reactions to schistosomes and eggs. These reactions in the liver were strikingly reduced in size and it was possible to observe several instances in which a parasite was in contact with normal tissue, virtually in the absence of any relevant sign of cell infiltration. Such a result was expected, in view of the well-documented identification of the schistosome granuloma with a thymus-dependent delayed hypersensitivity reaction (17). Local tissue reactions to schistosomes and eggs have recently been proposed to be responsible for certain resistance phenomena observed in the mouse (18). Therefore, the possibility could be considered that the suppression of resistance to reinfection in B rats was brought about-at least in part-by a suppression of host tissue inflammatory reactions. Byram and von Lichtenberg (19) have reported an analogous suppression of granuloma formation in S. mansoni-infected nude mice. In these animals, areas of hepatocellular damage were observed around schistosome ova and the authors suggested that this damage may result from the absence of a granulomatous response which normally contributes to the containment and processing of schistosome egg products. We have not observed similar parenchymal alterations in our B rats. In fact, liver cells surrounding schistosome eggs showed the same normal appearance as the hepatocytes in the proximity of adult worms (Figs. 3C and D). It should be recalled, however, that schistosome oviposition is severely impaired in the rat and that a complete maturation of eggs to the secretory stage does not usually occur in this nonpermissive host.

In conclusion, the extensive immunosuppression achieved in B rats permits the overall attribution of resistance to reinfection in this host to thymus-dependent immune phenomena, but does not allow a precise dissection of the mechanisms involved, due to the wide range of immune parameters which are affected in this

FIG. 3. Liver sections of normal and B rats infected 10 weeks with 250cercariae. (A) Intact rat; cellular infiltrates subverting the hepatic structure. Number sign indicates a large granuloma. Arrows point to infiltrated cells and bands of connective tissue dividing the hepatic parenchyma into irregular areas of variable size. (B) Intact rat; cellular infiltrates surrounding an egg. (A) x 100; (B) x400. (C) B rat; intact hepatic structure with a parasite (boxed area). (D) Boxed area at a higher magnification. (C) x 100; (D) x400.

CIOLI ET AL.

Schistosoma REINFECTION IN THYMECTOMIZED RATS 255

256 CIOLI ET AL.

experimental model (direct T-cell reactions, antibody production, eosinophilia, and tissue responses, among others).

An ancillary finding which has emerged from the present study consists of the observation that different rat strains may exhibit quite different levels of resistance to S. mansoni reinfection. Similar strain variations have been observed in hamsters (20) and in mice (Dean, personal communication) and may represent a potentially important clue for a better understanding of the mechanisms of resistance in various host species.

ACKNOWLEDGMENTS

This investigation received financial support from the Rockefeller Foundation (to D.C.) and from the Edna McConnel Clark Foundation (to G.D.).

The expert technical assistance of Roland0 Moroni, Roberto Hernandez, and Jeffrey Kouba is gratefully acknowledged.

REFERENCES

1. Cioli, D., Knopf, P. M., and Senft, A. W., Int. J. Parasitol. 7, 293, 1977. 2. Cioli, D., and Dennert, G., J. Immunol. 117, 59, 1976. 3. Cioli, D., Blum, K., and Ruppel, A., Exp. Parasitol. 45, 74, 1978. 4. Smithers, S. R., and Terry, R. J., Parasitology 55, 711, 196.5. 5. Phillips, S. M., Reid, W. A., and Sadun, E. H., Cell. Immunol. 28, 75, 1977. 6. Knopf, P. M., Nutman, T., and Reasoner, J., Exp. Parasitol. 41, 74, 1977. 7. Dennert, G., and Lotan, R., Eur. J. Immunol. 8, 23, 1978. 8. Cioli, D., Znt. J. Parasitol. 6, 349, 1976. 9. Phillips, S. M., Reid, W. A., Bruce, J. J., Hedlund, K., Colvin, R. D., Campbell, R., Diggs, C. L.,

and Sadun, E. H., Cell Immunol. 19, 99, 1975. 10. Butterworth, A. E., Curr. Top. Microbial. Immunol. 77, 127, 1977. 11. Knopf, P. M., Exp. Parasitol. 47, 232, 1979. 12. Basten, A., and Beeson, P. B., J. Exp. Med. 131, 1288, 1970. 13. Buchanan, R. D., Fine, D. P., and Colley, D. G., Amer. J. Pathol. 71, 207, 1973. 14. Hsu, C. K., Whitney, R. A., and Hansen, C. T., Nature (London) 262, 397, 1976. 15. Phillips, S. M., DiConza, J. J., Gold, J. A., and Reid, W. A., J. Immunol. 118, 594, 1977. 16. Knopf, P. M., and Cioli, D., Int. J. Parusitol. 10, 13, 1980. 17. Warren, K. S., Domingo, E. O., and Cowan, R. B. T., Amer. J. Pathol. 51, 735, 1967. 18. Dean, D. A., Minard, P., Murrell, K. D., and Vannier, W. E., Amer. J. Trop. Med. Hyg. 27,957,

1978. 19. Byram, J. E., and von Lichtenberg, F., Amer. .I. Trop. Med. Hyg. 26, 944, 1977. 20. Smith, M. A., and Clegg, J. A., Parasitology 73, 47, 1976.